The emphasis of the proposed research will be to continue the studies of the membrane enzyme, pyruvate oxidase. Enzymological and structural studies of the purified protein will be continued. In addition, a project directed at elucidating the nature of the interaction of the pure enzyme with the E. coli inner membrane will be initiated. Pyruvate oxidase is a flavoprotein dehydrogenase which feeds electrons into the respiratory chain of aerobically grown E. coli. Since the redox state of the bound flavin is an important factor in determining the affinity of the protein for the membrane, the properties of the flavin will be one focus of investigation. Optical spectroscopy and the use of electrochemical techniques will be emphasized. Chemical modification studies will be continued in our effort to determine which residues are required for catalytic activity and/or membrane binding. The use of sulfhydril-directed reagents will be a major focus of this work. A major effort will also be made to isolate peptides involved in membrane binding by using hydrophobic protein modification reagents in the presence of detergents and phospholipid bilayers. The nature of the mode of coupling of pyruvate oxidase to the membrane-bound components of the respiratory chain will be investigated. In addition, the respiratory chain itself will be biochemically fractionated and characterized using biochemical, biophysical, and immunological techniques.